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Identi Cation of Different Proteins Binding to Na, K-Atpase Α1 In Identication of Different Proteins Binding to Na, K-ATPase α1 in Lipopolysaccharide-Induced Acute Respiratory Distress Syndrome Cell Model by Proteomic Analysis Xu-Peng Wen Transplantation Center, the Third Xiangya Hospital, Central South University, Changsha, 410013, Hunan, China Yue-Zhong Zhang Clinical Medicine, Xiangya School of Medicine, Central South University, Changsha, 410083, China He Huang Hunan International Travel Health Care Center Tao-Hua Liu Clinical Medicine, Xiangya School of Medicine, Central South University, Changsha, 410083, China Qi-Quan Wan ( [email protected] ) Transplantation Center, the Third Xiangya Hospital, Central South University, Changsha, 410013, Hunan, China Research Article Keywords: ARDS, lipopolysaccharide, proteomics, Na, K-ATPase α1, A549 cell. Posted Date: July 9th, 2021 DOI: https://doi.org/10.21203/rs.3.rs-687826/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/19 Abstract Acute respiratory distress syndrome (ARDS) is characterized by refractory hypoxemia caused by accumulation of pulmonary uid, which is related to inammatory cell inltration, impaired tight junction of pulmonary epithelium and impaired Na, K-ATPase function, especially Na, K-ATPase α1 subunit. Up until now, the pathogenic mechanism at the level of protein during lipopolysaccharide- (LPS-) induced ARDS remains unclear. Using an unbiased, discovery and quantitative proteomic approach, the discovery of differentially expressed proteins binding to Na, K-ATPase α1 between LPS-induced A549 cell and control-A549 group is of particular interest for the current study. These proteins may help the clinical diagnosis and facilitate the personalized treatment of ARDS. We screened these Na, K-ATPase α1 interacting proteins, carried out the related Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, and found evident phenomena of ubiquitination and deubiquitination, as well as the pathways related to autophagy. We also chose some of the differentiated expressing proteins with signicant performance for further verication by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Among proteins with rich abundance, there were several intriguing ones, including the deubiquitinase (OTUB1), the tight junction protein zonula occludens-1 (ZO-1), the scaffold protein in CUL4B-RING ubiquitin ligase (CRL4B) complexes (CUL4B) and the autophagy-related protein sequestosome-1 (SQSTM1). Protein-protein interaction network showed that there were 244 signicantly enriched co-expression among 60 proteins in the group control-A549. while the group LPS-A549 showed 43 signicant enriched interactions among 29 proteins. In conclusion, our quantitative discovery-based proteomic approach identied commonalities, and revealed targets related to the occurrence and development of ARDS, being the rst study to investigate signicant differences in Na, K-ATPase α1 interacting proteins between LPS-induced ARDS cell model and control-A549 cell. 1. Introduction Acute respiratory distress syndrome (ARDS) is a potentially fatal clinical syndrome that occurs as a result of diversied pulmonary and extrapulmonary factors, characterized by excessive lung inammatory response, impaired tight junction of pulmonary epithelium, decreased pulmonary gas exchange ability and reduced alveolar uid clearance (AFC) of the lungs with consequent refractory hypoxemia.1 Every year, over 3 million patients worldwide suffer from ARDS. 10% of intensive care unit (ICU) patients were admitted as a result of ARDS. With high morbidity and mortality, it is a hot spot in the eld of intensive care and respiratory medicine.2 Effective removal of excess edema uid in the alveoli and maintenance of dry alveolar space are the main ways to relieve ARDS.3 Over the past decade, considerable work has been done for eliminating excessive accumulation of alveolar edema uid to relieve ARDS and is still in progress. The apically-located epithelial Na+ channel (ENaC) and sodium pump, namely Na, K-ATPase, on the basolateral surface of alveolar type epithelial cells (AT ) mediated sodium ion transport is the main dynamic of AFC.4–6 The imbalance of Na, K-ATPase will aggravate the formation of pulmonary edema by limiting Na+ transport and destroying the alveolar barrier function.7 Na, K-ATPase, is a ubiquitous enzyme consisting of three subunits. Among them, α-subunit plays a key role and is the most important one in sodium-water transport as the main driving force of Na+ and K+ exchange in the lung to promote uid clearance in the alveoli. There are four subtypes of α subunit, only α1 exists in lung.8 Na, K-ATPase α1 carries several binding domains and functional domains.9 Resolvin D1 can improve the expression of ENaC and Na, K- ATPase α1 protein and the activity of Na, K-ATPase enzyme through ALX/cAMP/PI3K pathway, thus improving the obstruction of pulmonary edema clearance caused by lipopolysaccharides (LPS).10 However, researchers have not explored the systemic regulation mechanism of Na, K-ATPase. Similarly, a study has reported that Maresin1 can enhance the protein expression of Na, K-ATPase α1, which may be achieved through the ALX/PI3K/AKT/Nedd4-2 pathway.6 Whereas, E3 ubiquitin ligase Nedd4-2 has no structural binding sites with Na, K-ATPase. Maresin1 could not increase Na, K-ATPase α1 level through increasing the phosphorylation of Nedd4-2 and its binding with 14-3-3 protein. The mechanism of Maresin1 regulating Na, K-ATPase α1 is not clear. Thus, investigating Na, K-ATPase α1-related pathway may provide new strategies and targets for ARDS treatment. But, a powerful tool to precisely and quantitatively detect changes in protein expression in response to ARDS is necessary. Unbiased discovery and the quantitative proteomic approach, enabling relatively comprehensive global analyses, have been used for identifying novel biomarkers and regulatory signal networks in lung diseases including ARDS.11–17 Studies identied some possible ARDS biomarkers, such as angiopoietin-2, surfactant proteins, glutathione, selectins, thrombomodulin, adenosine, Clara cell protein and many other biomarkers, which were reviewed before.11,12,15−18 Several clinical trials failed since there was more than one pathway causing ARDS.19–21 The research of ARDS biomarkers concerning several different elds, requiring a more comprehensive study method than traditional ones, thus non-targeted proteomics can be very suitable. In the present study, we utilized LPS-induced human AT II cell line (A549) as a model of ARDS,22–24 and detected the changes in the protein expression proles of LPS-A549 group compared with control-A549 group and control-IgG group. Among them, protein complexes and protein interaction networks are essential mediators of most biological functions. Currently, the majority of studies on the composition of protein complexes are carried out by anity purication mass spectrometry (AP-MS/MS), or by co-immunoprecipitation mass spectrometry (Co-IP-MS) for untransfected native samples, and present a static view of the system (Figs. 1 and 2) (free images were obtained from Aksomics). We employed anity purication (AP) or co-immunoprecipitation (Co-IP) technology to separate endogenous or labeled bait proteins and the proteins interacting with them. Then, we used liquid chromatography-tandem mass spectrometry (LC- MS/MS) technology to identify and quantify these proteins, combined with Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, constructing the protein interaction network. Altogether, differential protein expression data may provide a valuable resource to reveal potential molecular targets for ARDS treatment. 2 Materials And Methods 2.1. Reagents Page 2/19 LPS (Escherichia coli serotype 055: B5), TCEP (tris (2-carboxyethyl) phosphine), IAA (iodoacetamide), PBS, C18 columns (3M), FA (Formic acid, LC-MS), TFA (Triuoroacetic acid, HPLC) and 10% ammonium hydroxide were purchased from Sigma-Aldrich (St Louis, MO, USA); Na, K-ATPase α1 antibody was purchased from Proteintech, USA; ACN (Acetonitrile, LC-MS) and H2O (LC-MS) were purchased from J.T.Baker (PA, USA); Trypsin (sequence grade) was purchased from Promega (Madison, WI, USA); NP-40 (Nonidet P 40) and Ammonium bicarbonate (ABC) were purchased from Sangon Biotech (Shanghai, China); Dynabeads® Anti Rabbit IgG was purchased from Thermo Scientic (Rockford, USA); Normal rabbit IgG was purchased from Cell signaling technology (MA, USA); Protease inhibitor cocktail (PIC) was purchased from Kangchen Bio-tech (Shanghai, China). 2.2 Cell line and cell culture A549 cell line was purchased from ATCC; A549 cells were seeded into plastic culture dishes at 1×106/cm2 and cultured in a humidied incubator (21 % O2, 5 % CO2, 37°C) in DMEM with 10% fetal bovine serum (FBS), 2 mM L-glutamine, 100 units/ml penicillin, and 100µg/ml streptomycin. For all experiments, cells were grown and maintained in six-well plates, and cells were serum deprived for 24 h prior to pretreatment with LPS at a concentration of 1 ug/ml for 12 h at 37°C. 2.3 Sample preparation For all experiments, cells were grown and maintained in six-well plates without 10% FBS for 12 hours before the experiment started. Subsequent experiments were conducted on medium without 10% FBS. There were two groups: A: A549 cells, B: A549 cells + LPS (1µg/ml, cultured for 12h). ARDS was induced by LPS according to previous reports.10 The proteins were detected by mass spectrometry, and the amount of protein was4 mg. Na, K-ATPase α1 antibody was used to pull down the Na, K-ATPase α1 proteins in two groups of cells. These proteins were pulled down for label-free mass spectrometry to understand the binding and interacting protein, and then the protein of interest was selected for verication. 2.4 Sample lysis (1) Just before being used, Mix PBSN (PBS, 1% NP-40) and Protease inhibitor cocktail (PIC) were pre-chilled at 4°C. (2) Add 1mL lysis buffer to cell pellet, mix well, and sonicate to dissolve with ice bath. (3) 14000 g, centrifuge 15min at 4°C, transfer the supernatant to a new EP tube and keep it on ice.
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